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Advanced genomic technologies such as whole exome or whole genome sequencing have improved diagnoses and disease outcomes for individuals with genetic diseases. Yet, variants of unknown significance (VUS) require rigorous validation to establish disease causality or modification, or to exclude them from further analysis. Here, we describe a young individual of Polynesian ancestry who in the first 13 mo of life presented with SARS-CoV-2 pneumonia, severe enterovirus meningitis and adenovirus gastroenteritis, and severe adverse reaction to MMR vaccination. Genomic analysis identified a previously reported pathogenic homozygous variant in IFNAR1 (c.1156G > T, p.Glu386* LOF), which is common in Western Polynesia. Moreover, a new and putatively deleterious canonical splice site variant in DOCK8 was also found in homozygosity (c.3234 + 2T > C). This DOCK8 variant is common in Polynesians and other under-represented ancestries in large genomic databases. Despite in silico bioinformatic predictions, extensive in vitro and ex vivo analysis revealed the DOCK8 variant likely be neutral. Thus, our study reports a novel case of IFNAR1 deficiency, but also highlights the importance of functional validation of VUS, including those predicted to be deleterious, and the pressing need to expand our knowledge of the genomic architecture and landscape of under-represented populations and ancestries.

RNAs are remarkably versatile molecules that can fold into intricate three-dimensional (3D) structures to perform diverse cellular and viral functions. Despite their biological importance, relatively few RNA 3D structures have been solved, and our understanding of RNA structure-function relationships remains in its infancy. This limitation partly arises from challenges posed by RNA's complex conformational landscape, characterized by structural flexibility, formation of multiple states, and a propensity to misfold. Recently, cryoelectron microscopy (cryo-EM) has emerged as a powerful tool for the visualization of conformationally dynamic RNA- only 3D structures. However, RNA's characteristics continue to pose challenges. We discuss experimental methods developed to overcome these hurdles, including the engineering of modular modifications that facilitate the visualization of small RNAs, improve particle alignment, and validate structural models.

Purpose Transcription factor 3 (TCF3) encodes 2 transcription factors generated by alternative splicing, E12 and E47, which contribute to early lymphocyte differentiation. In humans, autosomal dominant (AD) E47 transcription factor deficiency is an inborn error of immunity characterized by B-cell deficiency and agammaglobulinemia. Only the recurrent de novo p.E555K pathogenic variant has been associated with this disease and acts via a dominant-negative (DN) mechanism. In this study, we describe the first Asian patient with agammaglobulinemia caused by the TCF3 p.E555K variant and provide insights into the structure and function of this variant. Methods TCF3 variant was identified by inborn errors of immunity-related gene panel sequencing. The variant E555K was characterized by alanine scanning of the E47 basic region and comprehensive mutational analysis focused on position 555. Results The patient was a 25-year-old male with B-cell deficiency, agammaglobulinemia, and mild facial dysmorphic features. We confirmed the diagnosis of AD E47 transcription factor deficiency by identifying a heterozygous missense variant, c.1663 G>A; p.E555K, in TCF3. Alanine scanning of the E47 basic region revealed the structural importance of position 555. Comprehensive mutational analysis focused on position 555 showed that only the glutamate-to-lysine substitution had a strong DN effect. 3D modeling demonstrated that this variant not only abolished hydrogen bonds involved in protein-DNA interactions, but also inverted the charge on the surface of the E47 protein. Conclusions Our study reveals the causative mutation hotspot in the TCF3 DN variant and highlights the weak negative selection associated with the TCF3 gene.

Purpose: After September 11, 2001, nuclear threat prompted government agencies to develop medical countermeasures to mitigate two syndromes, the hematopoietic-acute radiation syndrome (H-ARS) and the higher-dose gastrointestinal-acute radiation syndrome (GI-ARS), both lethal within weeks. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS, no mitigator potentially deliverable under mass casualty conditions preserves the GI tract. We recently reported that anti-ceramide single-chain variable fragment (scFv) mitigates GI-ARS lethality, abrogating ongoing small intestinal endothelial apoptosis to rescue Lgr5+ + stem cells. Here, we examine long-term consequences of prevention of acute GI-ARS lethality. Methods and Materials: For these studies, C57BL/6J male mice were treated with 15 Gy whole body irradiation, the 90% GIARS lethal dose for this mouse strain. Results: Mice irradiated with 15 Gy alone or with 15 Gy + bone marrow transplantation (BMT) or anti-ceramide scFv, succumb to an ARS within 8 to 10 days. Autopsies reveal only mice receiving anti-ceramide scFv at 24 hours post-whole body irradiation display small intestinal rescue. No marrow reconstitution occurs in any group with attendant undetectable circulating blood elements. Mice receiving 15 Gy + BMT + scFv, however, normalize blood counts by day 12, suggesting that scFv also improves marrow reconstitution, a concept for which we provide experimental support. We show that at 14 Gy, the upper limit dose for H-ARS lethality before transition to GI-ARS lethality, anti-ceramide scFv markedly improves marrow take, reducing the quantity of marrow-conferring survival by more than 3-fold. Consistent with these fi ndings, mice receiving 15 Gy + BMT + scFv exhibit prolonged survival. At day 90, before sacrifice, fi ce, they display normal appearance, behavior, and serum biochemistries, and surprisingly, at full autopsy, near-normal physiology in all 42 tissues examined. Conclusions: Anti-ceramide scFv mitigates GI-ARS lethality and improves marrow reconstitution rendering prolonged survival with near normal autopsies. (c) 2023 Elsevier Inc. All rights reserved.

RNA's ability to form and interconvert between multiple secondary and tertiary structures is critical to its functional versatility and the traditional view of RNA structures as static entities has shifted towards understanding them as dynamic conformational ensembles. In this review we discuss RNA structural ensembles and their dynamics, highlighting the concept of conformational energy landscapes as a unifying framework for understanding RNA processes such as folding, misfolding, conformational changes, and complex formation. Ongoing advancements in cryo-electron microscopy and chemical probing techniques are significantly enhancing our ability to investigate multiple structures adopted by conformationally dynamic RNAs, while traditional methods such as nuclear magnetic resonance spectroscopy continue to play a crucial role in providing high-resolution, quantitative spatial and temporal information. We discuss how these methods, when used synergistically, can provide a comprehensive understanding of RNA conformational ensembles, offering new insights into their regulatory functions.

Background Cryptococcosis is a life-threatening disease caused by Cryptococcus neoformans or C. gattii. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) in otherwise healthy adults with cryptococcal meningitis have been described since 2013. We searched for neutralizing auto-Abs in sera collected from Colombian patients with non-HIV-associated cryptococcosis in a retrospective national cohort from 1997 to 2016. Methods We reviewed clinical and laboratory records and assessed the presence of neutralizing auto-Abs against GM-CSF in 30 HIV negative adults with cryptococcosis (13 caused by C. gattii and 17 caused by C. neoformans). Results We detected neutralizing auto-Abs against GM-CSF in the sera of 10 out of 13 (77%) patients infected with C. gattii and one out of 17 (6%) patients infected with C. neoformans. Conclusions We report eleven Colombian patients diagnosed with cryptococcosis who had auto-Abs that neutralize GM-CSF. Among these patients, ten were infected with C. gattii and only one with C. neoformans.

Despite therapeutic efficacy observed with immune checkpoint blockade in advanced melanoma, many tumors do not respond to treatment, representing a need for new therapies. Here, we have generated chimeric antigen receptor (CAR) T cells targeting TYRP1, a melanoma differentiation antigen expressed on the surface of melanomas, including rare acral and uveal melanomas. TYRP1-targeted CART cells demonstrate antigen-specific activation and cytotoxic activity in vitro and in vivo against human melanomas independent of the MHC alleles and expression. In addition, the toxicity to pigmented normal tissues observed with T lymphocytes expressing TYRP1-targeted TCRs was not observed with TYRP1-targeted CAR T cells. Anti-TYRP1 CAR T cells provide a novel means to target advanced melanomas, serving as a platform for the development of similar novel therapeutic agents and as a tool to interrogate the immunobiology of melanomas.

Judy Lieberman is a professor of pediatrics and adjunct professor of genetics at Harvard Medical School and an endowed chair in cellular and molecular medicine. Her lab studies cytotoxic T lymphocytes (CTL), key cells in the immune defense against viral infection and cancer, as well as molecular pathways activated by the granzymes, and how RNA interference (RNAi) regulates cell differentiation in health and disease states. We spoke to Judy about advice for early career researchers, how she first become interested in cytotoxic T lymphocytes, and key people who have provided mentorship across her career.

Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs (Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity.

Streptococcus pneumoniae carriage studies are crucial to monitor changes induced by use of pneumococcal conjugate vaccines and inform vaccination policies. In this cross-sectional study, we examined changes within the pneumococcal population following introduction of PCV13 in 2015 in the National Immunization Program (NIP), in Portugal. In 2018-2020 (NIP-PCV13), we obtained 1450 nasopharyngeal samples from children <= 6 years attending day-care. We assessed serotypes, antimicrobial resistance, and genotypes (MLST and GPSC) and compared findings with earlier periods: 2009-2010 (pre-PCV13), 2011-2012 (early-PCV13), and 2015-2016 (late-PCV13). Pneumococcal carriage prevalence remained stable at 60.2 %. Carriage of PCV13 serotypes was 10.7 %, markedly reduced compared to pre-PCV13 period (47.6 %). The most prevalent PCV13 serotypes were 19F, 3, and 19A all showing a significant decreasing trend compared to the pre-PCV13 period (from 7.1 % to 4.7 %, 10.1 % to 1.8 %, and 14.1 % to 1.8 %, respectively), a notable observation given the described limited effectiveness of PCV13 against serotype 3. Non-vaccinated children and children aged 4-6 years were more likely to carry PCV13 serotypes (2.5-fold, 95 %CI [1.1-5.6], and 2.9-fold, 95 %CI [1.3-6.8], respectively). The most prevalent non-PCV13 serotypes were 15B/C, 11A, 23B, 23A, and NT, collectively accounting for 51.9 % of all isolates. In total, 30.5 % of all pneumococci were potentially covered by PCV20. Resistance to penicillin (lowlevel) and macrolides increased significantly, from 9.3 % and 13.4 %, respectively, in the late-PCV13 period, to approximately 20 % each, mostly due to lineages expressing non-PCV13 serotypes, nearing pre-PCV13 levels. An expansion of lineages traditionally associated with PCV13 serotypes, like CC156-GPSC6 (serotype 14) and CC193-GPSC11 (serotype 19F), but now predominantly expressing non-PCV13 serotypes (11A, 15B/C, and 24F for GPSC6; and 15A and 21 for GPSC11) was noted. These findings indicate that the pneumococcal population is adapting to the pressures conferred by PCV13 and antimicrobial use and indicate the need to maintain close surveillance.